Our analysis of a mouse mutation causing embryonic arrest prior to gastrulation led to the identification and isolation of nodal, a gene encoding a member of the TGF-beta superfamily of secreted signaling molecules. We found that injection of in vitro synthesized nodal mRNA into frog or fish embryos, or treatment of frog embryos with recombinant nodal protein, redirected cells to a mesodermal fate, suggesting that nodal acts as a mesoderm inducing factor during normal mouse development. We have also shown that nodal has an important role later in embryonic development, during the establishment of the left- right (L/R) body axis. All vertebrates have characteristic and conserved L/R visceral asymmetries, for example the left-sided heart. Recently, studies from several different labs have begun to explain how the embryonic L/R axis is established. We showed that nodal is one of a small number of genes expressed asymmetrically along the L/R axis in the chick embryo before the appearance of normal morphological L/R asymmetry. This asymmetric expression has been evolutionarily conserved: nodal is asymmetrically expressed in mouse embryos at similar stages and locations, as is Xenopus nodal related-1 (Xnr-1) in frog embryos. In further experiments in the chick, embryos were manipulated so that nodal was expressed symmetrically on both the left and right sides of the body, or so that nodal was not expressed at all. In both sets of experiments, approximately half the embryos developed a right-sided heart. These results have shown that nodal is not required for the heart to form, rather its asymmetric expression is necessary for the heart to develop with characteristic polarity. We examined nodal expression in the situs inversus viscerum (iv) mouse mutant in which L/R development is randomized (approximately half of iv mice have normal heart placement while half have reversed heart location). In individual iv embryos, nodal expression was found to be either symmetric (on both sides or on neither side), or asymmetric (on the left side only or on the right side only). Thus, mutations in the iv gene disturb L/R development by randomizing the location of nodal expression.More recently, we have been studying 4 additional mutants that show altered L/R development. These include mutations at the Ft, Mgat, nt and SIL loci. We analyzed nodal expression in all four strains and again found altered patterns: Mgat and nt were random like iv whereas all SIL and Ft embryos expressed nodal on both sides. Currently, we are analyzing iv, SIL and Mgat mutants in greater detail to understand why each affects nodal expression so differently. In contrast to iv, we found defects in embryonic structures along the middle of the embryo in Mgat and SIL mutants, but the timing and nature of the defects were different. A unique finding in SIL mutants was the lack of activity of the sonic hedgehog (shh) signal transduction pathway, an essential component of a number of developmental processes. We are now studying the role of SIL in the shh pathway and whether bilateral symmetry of nodal expression results from disrupted shh signaling, defective midline structures or both. These results further support the critical role nodal plays in interpreting and relaying L/R patterning information in vertebrates. - Animal models, cell proliferation, Development, embryonic stem cells, Genetics, growth factors, in situ hybridization, protein folding, receptors, - Neither Human Subjects nor Human Tissues